R

TYDAL

WIDAL ANTIGEN SET / ANTIGENS FOR SLIDE AND TUBE TESTS

INTENDED USE
TYDAL® is a Widal slide and tube agglutination test that detects the presence of the serum agglutinins (O, H) in the
patient’s serum, with typhoid and paratyphoid fever.

SUMMARY
Enteric fever occurs when pathogenic microorganisms like S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C
infect the human body. During the course of disease, the body responds to this antigenic stimulus by producing
antibodies whose titre rises slowly in early stages, to a maxima and then slowly falls till it is undetectable.
Antibodies to Salmonella organisms may be detected in the patient serum from the second week after onset of
infection. Information regarding the titres and whether or not they are rising or falling can be obtained by
performing serological tests using TYDAL® antigen suspensions. Usually tube titres of 1:80 and above are taken
as diagnostically significant, however for endemic areas higher cut-offs may need to be established.

REAGENT
TYDAL® contains ready to use concentrated, smooth antigen suspensions of the bacilli; S. typhi 'O', S. typhi
'H', S. paratyphi 'AO', S. paratyphi 'BO', S. paratyphi 'AH', S. paratyphi 'BH', S. paratyphi 'CH', S. paratyphi 'CO'
and / or polyspecific positive control reactive with these antigens.
Each batch of reagents undergoes rigorous quality control at various stages of manufacture for its specificity and
performance.

REAGENT STORAGE AND STABILITY

1. Store the reagents at 2-80C. DO NOT FREEZE .Keep the reagents away from direct sunlight.
2. The shelf life of reagents is as per the expiry date mentioned on the reagent vial labels. Do not use beyond
expiry date.
3. Once opened the shelf life of the reagent vial is as described on the reagent vial label provided it is not
contaminated.

PRESENTATION
4x5 ml 8x5 ml* 2x5 ml 2x5 ml 2x5 ml 2x2x 2x2x 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml
5 ml 5 ml
105200045

105200085

105210025

105200025

105220025

105210225

105200225

105220005

105230005
105240005

105250005

105280005

105260005
105270005
105290005

REF

Antigens O, H, O, H, O, H O, H O, H O, H O, H O H AO BO CO AH BH CH
AH, AH, BH,
BH CH, AO
BO, CO

Control + 0.4 ml 2.0 ml 0.4ml 0.4ml 0.4ml 0.4ml 0.4ml

Control - 2.0 ml 0.4ml 0.4ml 0.4ml
MIXING STICKS
LADDER 4 6 4 4 4
DISPENSER PP
TUBES 50 50 50 50 50
RUBBER TEAT 1 1 1 1 1
SLIDE 1 1 1 1 1
PACKAGE INSERT 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

* 8 x 5 ml pack is marketed as TYDAL® PLUS.

ADDITIONAL MATERIAL REQUIRED
Slide test method: Stop watch, Variable Micropipettes.
Quantitative method: Timer, Kahn tubes / test tubes, Pipettes (0.1ml, 1ml), Physiological saline, Incubator
(370C), Test tube rack.

PRINCIPLE
When the coloured, smooth, attenuated TYDAL® antigen suspensions are mixed / incubated with patient serum,
anti-Salmonella antibodies present in the patient serum react with the antigen suspensions to give agglutination.
Agglutination is a positive test result, indicating presence of anti-Salmonella antibodies in the patient serum.
No agglutination is a negative test result indicating absence of anti-Salmonella antibodies.

NOTE
1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
2. The S. typhi 'O', S. paratyphi 'CO' reagents contain 0.5% Phenol, S. typhi 'H', S. paratyphi 'AH', S. paratyphi
'BH', S. paratyphi 'CH' reagents contain 0.3% Formaldehyde and S. paratyphi 'AO', S. paratyphi 'BO' reagents
contain 0.7% Ethanol along with 0.1% Sodium azide as preservatives. Avoid contact with skin and mucosa.
Do not breathe vapour. In case of contact with eyes, rinse immediately with plenty of water and seek medical
advice. Sodium azide may react with lead and copper in plumbing and form highly explosive metal oxides, on
disposal flush with large quantities of water.
3. The reagent can be damaged due to microbial contamination or on exposure to extreme temperatures. It is
recommended that the performance of the reagent be verified with the positive and negative controls. Positive
control provided with the kit only for TYDAL® 4 x 5 ml set (REF.: 105200045), 2 x 5 ml set (REF.: 105210025 &
REF 105200025), 2 x 2 x 5 ml set (REF.: 105210225) and TYDAL® PLUS 8 x 5 ml set (REF.: 105200085).
Negative Control provided with the kit only for 8 x 5 ml (REF.: 105200085), 2 x 5 ml (REF 105200025) and
2 x 2 x 5 ml (REF 105200225).
4. Shake the reagent vials well before use to disperse the antigen suspension uniformly and improve test
readability.
5. Only clean and dry slides / tubes must be used. Clean the slide / tube with distilled water and dry.
6. TYDAL® antigen suspensions are not from human sources hence contamination due to HBsAg and HIV is
practically excluded.
7. Accessories provided with the kit only must be used for optimum results. (Applicable only for TYDAL® 2 x 2 x 5
ml set (REF.: 105210225, 105200225), 2 x 5 ml set (REF.: 105200025), 4 x 5 ml set REF.: 105200045) and
TYDAL® PLUS 8 x 5 ml set (REF.: 105200085).
8. Do not use damaged or leaking reagents.

SAMPLE COLLECTION AND STORAGE

1. No special preparation of the patient is required prior to sample collection by approved techniques. Do not use
haemolysed and turbid samples.
2. Clean and dry glassware free from detergents must be used for sample collection.
3. Do not heat inactivate the serum.
4. Though freshly collected serum is preferable, store samples at 2-80C in case of delay in testing, for upto 72
hours.

TEST PROCEDURE
Bring reagents and samples to room temperature before testing.
Tighten the cap of each; Plastic reagent bottle, positive control and negative control bottles respectively in the
clockwise direction to pierce the bottle nozzle.

Slide Screen Method

1. Place one drop of positive control onto a reaction circle of the slide.
2. Place 50 µl of physiological saline onto the next reaction circle of the slide.
3. Place one drop of patient’s serum to be tested onto each of the required number of reaction circles.
4. Add one drop of appropriate TYDAL® antigen suspension to the reaction circles containing Positive control &
physiological saline.
5. Add one drop of appropriate TYDAL® antigen suspensions to the reaction circles containing the patient’s
serum.
6. Mix contents of each circle uniformly over the entire circle with separate mixing sticks.
7. Rock the slide gently back and forth, and observe for agglutination macroscopically at one minute.

Slide Semi-Quantitative Method

1. Using a pipette place 80 µl, 40 µl, 20 µl, 10 µl, and 5 µl of patient serum to be tested on 5 different reaction
circles on the slide. The corresponding titres obtained will be 1:20, 1:40, 1:80, 1:160, & 1:320 respectively.
2. Follow step No. 5-7 of slide screen method.
Note: This method is recommended for obtaining quick approximate titres only.

Quantitative Method
Tube-test Procedure
1. Take appropriate number of sets (as required; one set for each antigen suspension) of 8 Kahn tubes / test
tubes and label them 1 to 8.
2. Pipette into tube No. 1 of all sets 1.9 ml of physiological saline.
3. To each of the remaining tubes (2 to 8) add 1 ml of physiological saline.
4. To tube No. 1 of all sets add 0.1 ml of serum sample to be tested and mix well.
5. Transfer 1 ml of the diluted serum sample from tube No. 1 to tube No. 2 and mix well.
6. Transfer 1 ml of the diluted serum sample from tube No. 2 to tube No. 3 and mix well. Continue this serial
dilution till tube No. 7 in each set.
7. Discard 1.0 ml of the diluted serum from tube No.7 of each set.
8. Now the dilutions of the serum sample achieved from tube No. 1 to 7 respectively in each set is as follows:
1:20, 1:40, 1:80, 1:160, 1: 320, 1:640, 1: 1280. Tube No. 8 in all the sets, serves as a saline control.
9. To all the tubes (1 to 8) of each set add one drop of the respective well-mixed TYDAL® antigen suspensions
from the reagent vials and mix well.
10. Cover and incubate at 370C overnight (approximately 18 hours).
11. Dislodge the sedimented button gently and observe for agglutination.

INTERPRETATION OF RESULTS
Slide Screen Method
Agglutination is a positive test result and indicates presence of the corresponding antibody in the patient’s serum.
No agglutination is a negative test result and indicates absence of the corresponding antibody in the patient
serum.
Slide Semi-Quantitative Method
Agglutination is a positive test result. The titre of the patient serum corresponds to the visible agglutination in the
test circle with the least amount of serum sample.
Quantitative Method
The titre of the patient serum using TYDAL® antigen suspensions is the highest dilution of the serum sample that
gives a visible agglutination.

REMARKS
1. Positive results obtained in the slide test should be confirmed with the tube test to establish whether the titres
are diagnostically significant or not.
2. TAB vaccinated patients may show a high titre of antibodies to each of the antigens. Similarly, an amnestic
response to other vaccines and unrelated fevers in case of patients who have had prior infection or
immunization may give a false result.
3. Agglutinins usually appear by the end of the first week of infection, blood sample taken earlier may give a
negative result.
4. A rising titre is more significant than a single high titre. It is therefore necessary to evaluate two or more serum
samples taken at 4- 6 days intervals after the onset of the disease.
5. 'O' being a somatic antigen brings about a coarse, compact, granular agglutination whereas 'H' being a
flagellar antigen brings about larger, loose, flocculant agglutination.
6. While the 'O' antigen is species specific, the 'H' antigen is specific to the serotype.
7. Serological findings are not intended as a substitute for culture. An appropriate attempt should be made to
recover and identify the etiologic organisms through various culture and biochemical tests.
8. Generally antibody titres of 1:80 or more are considered clinically and diagnostically significant. However the
significant titre may vary from population to population and needs to be established for each area.
 9. False positive results are likely if the test is read more than one minute after mixing on the slide test.
10. Any deviation in test procedure could result in variable results.
11. Since techniques and standardization vary from lab to lab one tube difference in tube titres can be expected.
12. Use a separate disposable tip for each sample to prevent cross contamination.
13. After usage the antigen suspension should be immediately recapped and replaced at 2-80C.
14. It is recommended that results of the tests should be correlated with clinical findings to arrive at the final
diagnosis.
15. The performance of the reagents should be validated occasionally using the positive control provided. Good
physiological saline may be used as a negative control.
16. Cap the reagent vial tightly after use to avoid spilling of reagent inside the cap.
17. Always keep the reagent vial in upright position during storage.

PERFORMANCE CHARACTERISTICS
1. The positive control antisera should produce 1+ or greater agglutination at 1: 80 in the slide and tube test
when tested with the TYDAL® antigen suspensions.
2. The negative control should show no agglutination with any of the TYDAL® antigen suspensions.
3. Generally accepted performance characteristic of this type of test is 70% specificity and sensitivity.
4. Reproducibility of TYDAL® antigen suspensions is 100% (+/- one double dilution).

WARRANTY
This product is designed to perform as described on the label and package insert. The manufacturer disclaims any
implied warranty of use and sale for any other purpose.

BIBILIOGRAPHY
(1). Cruickshank R., (1982), Medical Microbiology, 12th Edition, 403. (2).Felix A., (1942), Brit. Med. J., 11, 597-
600. (3). Data on file: Tulip Diagnostics (P) Ltd.

SYMBOL KEYS

Temperature Contains sufficient

Manufacturer
limitation for <n> tests

Consult
Use By Instructions This way up
for use

Date of Catalogue
Manufacture REF Number CONTROL + Positive control

Batch Number/ In vitro Diagnostic

LOT Lot Number IVD Medical Device CONTROL - Negative control

May be fatal if swallowed or enters airways

Harmful if inhaled, or if swallowed or
if in contact with skin
Danger Formaldehyde Authorised Representative
H350-H317
May cause irritation to skin and/or eyes
May cause irritation to the airways
EC REP in the European Community
P201;P281; P308+P313 and/or drowsiness or dizziness.
P280;P333+P313;P363 May cause an allergic skin reaction

Manufactured by
TULIP DIAGNOSTICS (P) LTD.
0520-9/0421/VER-05

Registered Office Manufacturing Unit

GITANJALI, TULIP BLOCK, DR. ANTONIO DO REGO BAGH, PLOT NO. UTILITY VIII, PHASE III B, VERNA IND.
ALTO SANTACRUZ, BAMBOLIM COMPLEX P.O., GOA-403 202, ESTATE, VERNA, GOA - 403 722, INDIA.
INDIA. Website: www.tulipgroup.com

EC REP
CMC Medical Devices & Drugs S.L., C/ Horacio Lengo No. 18, CP 29006, Malaga, Spain